Fluorescence resonance energy transfer imaging via fluorescence polarization measurement

Cohen-Kashi, Meir; Namer, Yaniv; Deutsch, Mordechai

doi:10.1117/1.2209955

Cited by 9 publications

(3 citation statements)

References 16 publications

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“…Methods used to detect the presence of FRET include those based on 1. intensity measurements with various steps to correct for spectral bleed-through 6,7 , 2. the acceptor photobleaching approach 8,9 , 3. spectral imaging microscopy to measure a complete per pixel emission spectrum, [10][11][12] 4. fluorescence polarization anisotropy measurements that compare the orientation of excited and emitting molecules, [13][14][15][16] and 5. monitoring changes in donor or acceptor fluorescence lifetimes either in the time domain [17][18][19][20] or in the frequency domain 9,18,[21][22][23] using fluorescence lifetime microscopy (FLIM).…”

Section: Introductionmentioning

confidence: 99%

Methodological considerations for global analysis of cellular FLIM/FRET measurements

et al. 2012

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Abstract. Global algorithms can improve the analysis of fluorescence energy transfer (FRET) measurement based on fluorescence lifetime microscopy. However, global analysis of FRET data is also susceptible to experimental artifacts. This work examines several common artifacts and suggests remedial experimental protocols. Specifically, we examined the accuracy of different methods for instrument response extraction and propose an adaptive method based on the mean lifetime of fluorescent proteins. We further examined the effects of image segmentation and a priori constraints on the accuracy of lifetime extraction. Methods to test the applicability of global analysis on cellular data are proposed and demonstrated. The accuracy of global fitting degrades with lower photon count. By systematically tracking the effect of the minimum photon count on lifetime and FRET prefactors when carrying out global analysis, we demonstrate a correction procedure to recover the correct FRET parameters, allowing us to obtain protein interaction information even in dim cellular regions with photon counts as low as 100 per decay curve.

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Section: Introductionmentioning

confidence: 99%

Methodological considerations for global analysis of cellular FLIM/FRET measurements

et al. 2012

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“…This phenomenon always causes enhancements of long-wavelength fluorescence and quenching of short-wavelength fluorescence. [16][17][18] Thus, the utilization of fluorescence encoding might produce cross talk with the label fluorescence, causing interference in both qualitative and quantitative analyses.…”

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confidence: 99%

Optical demodulation system for digitally encoded suspension array in fluoroimmunoassay

et al. 2017

J. Biomed. Opt

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Abstract. A laser-induced breakdown spectroscopy and fluorescence spectroscopy-coupled optical system is reported to demodulate digitally encoded suspension array in fluoroimmunoassay. It takes advantage of the plasma emissions of assembled elemental materials to digitally decode the suspension array, providing a more stable and accurate recognition to target biomolecules. By separating the decoding procedure of suspension array and adsorption quantity calculation of biomolecules into two independent channels, the cross talk between decoding and label signals in traditional methods had been successfully avoided, which promoted the accuracy of both processes and realized more sensitive quantitative detection of target biomolecules. We carried a multiplexed detection of several types of anti-IgG to verify the quantitative analysis performance of the system. A limit of detection of 1.48 × 10 −10 M was achieved, demonstrating the detection sensitivity of the optical demodulation system. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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“…There are two opposing trends: On the one hand, since the donor lifetime is shortened, the fluorophore has less time for rotational diffusion, thus the donor emission is more polarized. This can be used to calculate the FRET efficiency as [26]:…”

Section: Fluorescence Anisotropy-based Methodsmentioning

confidence: 99%

STED Microscopy of FRET Pairs

Loidolt-Krüger¹

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Förster resonance energy transfer (FRET) is a popular tool in life sciences, for example to detect protein-protein interactions and ligand binding, or to construct fluorescent biosensors for metabolites or ions. Obtaining such functional information provided by FRET from diffraction-unlimited images would be advantageous, because, for one thing, the spatial averaging of fluorescence signals could be reduced with a smaller detection volume, and, for another thing, FRET signals from neighboring subdiffraction areas could be distinguished. Thus, the goal of this thesis was to investigate the feasibility of measuring FRET using stimulated emission depletion (STED) microscopy.Numerical simulations of a single FRET pair under continuous-wave and pulsed STED illumination were performed to study the interplay of FRET and STED photophysics, including the influence of STED intensity and pulse delay. Große spektrale Verschiebungen der Fluoreszenz beider Fluorophore wurden in Einzelmolekülmessungen beobachtet. In STED-Bildern zeigte sich, dass eine Verschiebung der Emission hin zu kürzeren Wellenlängen die STED-Effizienz verringern kann und damit auch die Auflösung der Bilder. Die spektralen Verschiebungen ändern auch das spektrale Überlappintegral von Donoremission und Akzeptorabsorption und dadurch die FRET-Effizienz; die Größe dieses Effekts wurde abgeschätzt. vi Kurz zusammengefasst wurde ein photophysikalisches Modell für die Interferenz von STED und FRET eingeführt. Experimentelle Voraussetzungen für die STED-Mikroskopie von FRET-Paaren wurden identifiziert und eine adäquate Datenanalysestrategie wurde vorgeschlagen. Zusätzlich wurde die Photoumwandlung organischer Fluorophore charakterisiert und deren Auswirkung auf STED und FRET untersucht. vii 22 Effect of spectral shifts on the FRET efficiency

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Fluorescence resonance energy transfer imaging via fluorescence polarization measurement

Cited by 9 publications

References 16 publications

Methodological considerations for global analysis of cellular FLIM/FRET measurements

Methodological considerations for global analysis of cellular FLIM/FRET measurements

Optical demodulation system for digitally encoded suspension array in fluoroimmunoassay

STED Microscopy of FRET Pairs

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